Acyl-CoA synthetases (ACS) occupy a central position in fatty acid metabolism. For incorporation into phospholipids, triglycerides, or cholesterol esters, elongation, desaturation, degradation by b-oxidation, or acylation of proteins, a fatty acid must first be activated to its CoA thioester. Despite this vital function, only recently have the precise roles in metabolism of the numerous mammalian ACSs begun to be investigated. We have grouped ACSs by amino acid sequence similarity into at least six families. Two human and mouse ACS families, the very long-chain ACS (VLACS) family and a recently discovered ACS family that includes the BG gene (mutated in the fruit fly mutant "bubblegum") contain enzymes capable of activating very long-chain fatty acids (VLCFA; containing >22 carbons). VLCFA are essential components of complex lipids, particularly in the brain, and maintenance of VLCFA homeostasis is critical to normal health. Thus, enzymes that control the fate of VLCFA by any of the pathways noted above are of central importance. VLCFA accumulate in plasma and tissues of patients with X-linked adrenoleukodystrophy (XALD), a severe, often fatal neurodegenerative disorder. Defective VLCFA degradation via peroxisomal b-oxidation and decreased VLACS activity in peroxisomes have been implicated in the biochemical pathology of XALD. However, the gene defective in XALD, ABCD1, encodes ALDP, a peroxisomal protein of the ATP-binding cassette transmembrane transporter superfamily that has no VLACS activity. The VLACS and BG families contain a total of 8 enzymes. To understand overall VLCFA homeostasis, we must therefore understand the specific function of each of these enzymes. Therefore, we propose: 1) to develop a novel and generally applicable ACS assay, 2) to investigate the specific biological roles of human VLACSs, and 3) to characterize the BG1 knockout mouse and to assess the role of BG1 in XALD. Results of these studies will have a significant impact on our understanding of how fatty acids are channeled into specific metabolic pathways. Furthermore, they will facilitate elucidation of the role of enzymes belonging to the VLACS or BG families in XALD. [unreadable] [unreadable]